Mechanism of action of isopentenyl pyrophosphate isomerase: evidence for a carbonium ion intermediate

Abstract

Isopentenyl pyrophosphate isomerase catalyzes the interconversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. The isomerase from yeast has been purified to near homogeneity (purity > 90%). The substrate analogue (Z)-3-(trifluoromethyl)-2-butenyl pyrophosphate reacts at < 1.8 .times. 10-6 times the rate of dimethylallyl pyrophosphate. The enzyme is irreversibly inactivated by 2-(dimethylamino)ethyl pyrophosphate (I). These observations are consistent with a carbonium ion mechanism for the isomerization. Compound I is an analogue of the intermediate carbonium ion and probably acts as a transition state analogue. For I, kon'' = 2.1 .times. 106 M-1 min-1. No off-rate was detected and, therefore, Ki < 1.4 .times. 10-11 M. Upon denaturation of the inactivated enzyme, I is released unchanged. 2-(Trimethylammonio)ethyl pyrophosphate also inhibits with Ki'' = 7 .times. 10-7 M, kon'' = 4.4 .times. 104 M-1 min-1, and koff = 0.03 min-1. Substrate analogues without a positively charged nitrogen were relatively poor inhibitors. The best inhibitor of these is ethyl pyrophosphate, Ki = 10-4 M. The enzyme is inactivated by sulfhydryl-selective reagents. These reagents also prevent binding of I to the enzyme. The inactivation by iodoacetamide is dependent upon one ionizable group (pK = 9.3). The pH dependence of V and V/K for the isomerase-catalyzed reaction also depends upon a group with pK = 9.3.